Regulation of coagulation is essential for animal survival: deficiencies in coagulation, such as those associated with hemophilia, can produce life-threatening hemorrhage events, whereas excessive coagulation can lead to the formation of dangerous blood clots or can contribute to deleterious processes such as atherosclerosis. Clearly, the ability to modulate the extent of coagulation in cells, e.g., in endothelial cells, within a patient would provide a powerful tool for the treatment or prevention of a large number of common human diseases and conditions.
One key, rate limiting factor in the coagulation pathway is the generation of active thrombin. Indeed, tissue factor (TF)-dependent thrombin generation plays a critical role in hemostasis after tissue injury and also in the pathogenesis of multiple thrombotic disorders associated with a wide range of diseases, including cardiovascular disease, sepsis, and cancer. Accordingly, regulation of thrombin is critical for the prevention of thromboembolic disease and is dependent on an intricate balance between coagulation factors and inhibitors present in plasma and on the surface of cells. Thrombin generation in vivo occurs predominantly on cell surfaces and involves the interaction of tissue factor (TF), an integral membrane glycoprotein, with factor VII/VIIa to initiate blood coagulation. TF is present on the cell surface of lipid-laden macrophages (foam cells) and smooth muscle cells within human atherosclerotic plaques, and contributes to the thrombogenicity of the ruptured plaque. The generation of thrombin is subsequently regulated at the cell surface by thrombomodulin (TM), a cell surface receptor which promotes thrombin-dependent activation of the anticoagulant protein C pathway. Alterations in cell surface levels and/or activity of TF or TM can enhance thrombin generation, thereby increasing the risk of thromboembolic disease. Accordingly, a decrease in TF levels or activity, or an increase in TM levels or activity, would decrease the potential for thrombosis.
The biosynthesis and maturation of TF and TM, as well as other coagulation factors and inhibitors, occurs within the cellular secretory pathway which consists of the ER, Golgi complex and a variety of intermediate transport vesicles. The ER is the cellular organelle where these factors and inhibitors undergo a series of modifications, including folding, proteolytic processing, glycosylation, disulfide-bond formation and oligomeric assembly. Failure to fold properly or to assemble into correct oligomeric complexes can result in the generation of dysfunctional molecules and/or the retention of these molecules in the ER, followed by degradation.
To assist in the proper folding and assembly of newly synthesized proteins, the ER lumen contains a number of molecular chaperones, including GRP78/BiP, the 94 kDa glucose-regulated protein (GRP94), Calnexin, Calreticulin and ERp72. These molecular chaperones are important components of the quality control system of the ER that allows export of correctly folded and assembled proteins.
Overexpression of GRP78/BiP in cultured mammalian cells can affect the processing and secretion of several coagulation and fibrinolytic factors, including von Willebrand factor (vWf), factor VIII and tissue plasminogen activator (tPA). Recent studies using cultured human vascular endothelial cells have also shown that homocysteine, a thiol-containing amino acid implicated in the progression of atherothrombosis, induces the expression of GRP78/BiP while decreasing the processing and secretion of vWf.